Amide bond formation is one of the most studied reactions in chemistry and biology. It allows peptide and protein synthesis, and enables the synthesis of peptide-like molecules, known as peptidomimetic compounds, which are widely used in drug design and discovery programs. A plethora of reagents and reaction conditions have been developed over the years that facilitate amide bond formation by activating a carboxylic acid and mixing it with a primary or secondary amine. In a number of cases however the acylation reaction may not go to completion or may not proceed at all. Despite the progress and extensive research efforts in this field, so-called “difficult” amide bonds still exist that prevent access to a large number of compounds of great interest to the research community. In particular these include small cyclic peptides, large peptides and proteins and difficult peptide sequences. In these cases, attempts to force the acylation by heating or by increasing the activity of the activated ester result in undesirable side reactions, such as racemisation, or dimerisation. In these cases a different approach is required to facilitate the amide bond formation. In the past 10 years a number of auxiliary strategies have been developed that make use of an intramolecular acyl transfer to overcome some of these problems. These strategies and their targets are outlined in more detail below.
1. Native Ligation Chemistry
The general idea of chemical ligation is to synthesise large proteins in high purity. The process capitalises on the ability to generate highly homogeneous linear peptides of up to 50 residues long by using optimised solid phase peptide synthesis. These peptide segments are then linked or ligated in solution, using mutually reactive entities at the end of each segment. The major limitation to the existing ligation strategies is that they only work for a very limited number of ligation sites.
Several examples have been published where mutually reactive groups generate an amide isostere. In these first examples the ligation chemistry produced a modification in the peptide backbone of the product. In 1986, Kemp et al (1986) proposed a thiol-capture strategy, which is illustrated in Scheme 1.

Here two peptide segments are ligated using a mercaptobenzofuran substituent at the C-terminus of the first segment. The second segment, carrying a cysteine residue at the N-terminus, reacts to form a disulfide link. Following an O-to-N-acyl shift the disulfide link is cleaved, generating a “native” amide bond. This auxiliary strategy, although revolutionary in its own right, lacks versatility and has only been used successfully in a very limited number of cases (Fotouhi et al, 1989) due to the inherent difficulties in synthesising the selectively-protected peptide segments. It differs from our invention in the strategic approach and the design of the auxiliary. The same group has studied a number of parameters that influence the rate of the intramolecular acyl transfer, mostly focussing on the shape of the auxiliary (Kemp et al, 1981). This work is very different from our invention, and in no way suggested anything that is described in this invention.
In 1994 Dawson et al (1994) introduced the concept of native ligation, which allows the generation of proteins with a native or unmodified backbone from fully unprotected building blocks. This approach, outlined in Scheme 2, uses chemistry first described by Wieland for reacting amino acids.

In a first step an unprotected peptide-α-thioester selectively reacts with the thiol functionality on the N-terminal cysteine side chain of a second unprotected peptide. The initially-formed thioester undergoes a spontaneous acyl transfer in the aqueous buffer from a sulfur to a nitrogen atom, thereby generating a standard peptide bond. Several examples illustrate the significance of this work in enabling the synthesis of large proteins in high purity (Hackeng et al, 1997). One of the limitations of this native ligation strategy is that it relies on the presence of a cysteine residue somewhere in the middle of the target peptide sequence.
In an extension of this work, Canne et al (1996) reported a native ligation strategy that uses an auxiliary at the N-terminus of one of the peptide segments. The strategy avoids the necessity for an N-terminal cysteine residue, and expands the range of sites amenable to native chemical ligation to X-Gly and Gly-X. The strategy is outlined in Scheme 3.

A peptide-α-thioester reacts with an Nα (oxyethanethiol) peptide to produce the ligated product. The thioester-linked intermediate rearranges via an acyl transfer to an amide-linked product. The N—O bond at the tertiary amide can be readily cleaved using zinc dust in acidic aqueous solution, thereby releasing the oxyethanethiol auxiliary and producing the native backbone structure in the ligated product. The scope and limitation of this auxiliary approach was examined by selecting a range of different ligation sites.
It was found that the S-to-N acyl transfer only proceeded well for Gly-Gly ligation sites, but was more difficult when steric hindrance around the ligation site was increased. For example, in the case of a Gly-Ala ligation site the rearrangement, was incomplete after 10 hours in the pH 7.5 buffer. Lowering the pH to 4.5 accelerated the rearrangement, which was complete after 10 hours. In the case of a Phe-Gly ligation site rearrangement was almost complete after 2 days at 37° C. For the more hindered Phe-Ala ligation site no S-to-N acyl transfer step was observed, even after lowering the pH or leaving the sample at 37° C. for 24 hours. It was thus concluded that only Gly-X or X-Gly ligation sites will produce the target product. The limitation lies in the acyl-transfer step, which does not proceed for hindered ligation sites. The strategy differs from our invention in the way the auxiliary is introduced and removed, and in the design of the auxiliary.
Native ligations have also been performed using resin-bound peptides. One such strategy (Camarero et al, 1998) involves the assembly of a first peptide segment linked via a C-terminal thioester to the solid support, then adding the second segment containing a cysteine residue at the N-terminus, and performing the native ligation steps as for solution phase ligation. This has the advantage that handling of the intermediates is significantly reduced. Furthermore, several ligations can be performed in series using the same chemical approach. The limitations for the solid phase approaches are the same as for the solution phase chemistry, ie native ligation can only be performed at X-Cys, Gly-X or X-Gly sites.
2. Small Cyclic Peptides
Proteins and peptides are the primary means of initiating biological processes by interacting with macromolecular receptors. The crucial information determining the specific activity is often contained in relatively small sequences at the surface, and is determined by the three-dimensional conformation in which that sequence positions its side chains when interacting with the receptor. In the linear form, bioactive peptides can assume millions of different conformations, only very few of which are able to bind to the target receptor. In order to assess the important structural and dynamic properties that are critical to the biological potency and selectivity, conformational constraints are introduced, typically through cyclisation. Such cyclic molecules exist in more defined conformations, and are therefore very appealing from the point of view of pharmaceutical lead discovery. If activity is maintained or enhanced in these cyclic peptides, structural information is obtained, for example by NMR, X-ray or molecular modelling, and used to guide the development of therapeutic drugs. In addition, cyclisation generally promotes an increase of metabolic stability and bioavailability of peptides.
As the side chains are considered the main mediators for receptor interaction, cyclisation is preferably accomplished between the C- and N-termini. Whereas the synthesis of linear peptides generally proceeds well, head-to-tail cyclisation is often troublesome. This is particularly so for small peptides, ie. those less than seven residues long. All-L cyclic tetrapeptides for instance are not very accessible (Schmidt and Langner, 1997). The primary reason for ineffective cyclisation originates from what are called “difficult sequences”. In cyclisation terms this refers to a sequence-related inefficiency in “bringing the ends together” for head-to-tail cyclisation. Peptide bonds have strong π-character, and preferentially adopt a trans conformation. Linear precursors are therefore generally extended in conformation with terminal carboxylic acid and amine functional groups in remote positions, and are thus unfavourable for cyclisation. The problem is most prominent in the synthesis of small cyclic peptides, where activation of the C-terminus often results in the formation of linear and cyclic dimers or oligomers with low or no yield of target cyclic monomer.
There have been very few studies that address the “difficult” cyclisation issue. Cavalier-Frontin et al (1993) reported on the use of reversible chemical modifications of the peptide backbone to enhance cis-amide conformations. In the synthesis of cyclo-[Phe-Phe-Phe-Phe] (SEQ ID NO:32), each amide N was substituted with a BOC protecting group. The cyclisation yield increased from 1% to 27%. Similarly, the use of the N-(2-hydroxy-4-methoxybenzyl) (Hmb) group as a reversible N-backbone amide substituent has resulted in increases in yield of cyclic peptides (Ehrlich et al, 1996). It must be emphasised that here the “auxiliary” is placed on the backbone amide, and not on the N-terminal amine that reacts to form the “difficult” amide bond.
In the past two to three years a few studies where ligation chemistry was used in an intramolecular fashion have been reported. In these examples an initially larger ring is formed, and ring contraction accomplished through an intramolecular O-to-N or S-to-N acyl transfer.
In a first method reported by Botti et al (1996), linear unprotected peptides carrying a cysteine residue at the N-terminus and an aldehyde at the C-terminus, were cyclised to generate a thiazolidine containing cyclic peptide, as shown in Scheme 4. Initially a larger cycle is formed, in which the C- and N-termini are prepositioned for O-to-N acyl transfer and ring contraction to a smaller cycle. The disadvantage of this method is that the cyclic product always contains a thiazolidine moiety in the cycle, with an additional chiral centre which results in the formation of two diastereomers, and requires a cysteine residue at the N-terminus of the linear precursor. The method does not allow the generation of unmodified cyclic peptides, and is not a versatile procedure suitable for a combinatorial library approach.

Muir et al demonstrated that “native” ligation, using a cysteine residue at the N-terminus and a thioester at the C-terminus, can be applied in an intramolecular way to generate cyclic peptides (Camarero and Muir, 1997), as shown in Scheme 5. In Scheme 5, YAVTGRGDSPAASS is SEQ ID NO:33 and cyclo-CYAVTGRGDSPAASSG is SEQ ID NO:34.

A 15-residue unprotected peptide containing a C-terminal thioacid was converted to the head-to-tail cyclic peptide by dissolving the activated Cα-thioester in a pH 7.5 buffer. Cyclisation was complete in 10 minutes. The initially formed cyclic thioester rearranges quickly to form the final peptide bond. The strategy is not generic, as it requires a cysteine residue at the N-terminus.
In a similar way Shao et al (1998) showed that N-(oxyethanethiol)-glycine at the N-terminus can be employed to achieve cyclisation by allowing the thiol functionality to react regioselectively with a thioester at the C-terminus. These strategies, as the authors point out, are limited by the types of residues involved at the C- and N-termini. Cyclisation is only possible between Gly-X, where X is a non β-substituted residue. The slow acyl transfer is again the limiting factor in this cyclisation strategy.
None of these methods provides a versatile synthetic route to enable synthesis of cyclic peptides with unmodified or native peptide backbone. The first two methods require the presence of cysteine at the N-terminus and the last lacks versatility, as the ring contraction only proceeds for non-hindered cases. We have found that the latter approach does not provide access to a number of known “difficult” cyclic peptides.
3. Backbone Substitution
One of the major problems in solid phase peptide synthesis (SPPS) is the inefficient assembly of the so-called “difficult” sequences. Moreover, these sequence-related difficulties are impossible to predict a priori. The problems are believed to be mainly due to inter- and intra-chain aggregation during the assembly of the protected peptide on the solid support. This has led to the development of the backbone substitution strategy (Hyde et al, 1994) outlined below in Scheme 6. A 2-hydroxy-4-methoxybenzyl substituent (=Hmb) is introduced by using N,O-bis-Fmoc-protected (Hmb)-amino acids. In general acylation of N-substituted amino acids other than glycine requires forcing conditions, due to the massive steric hindrance imposed by the N-substituent. In the case of the Hmb-substituted amino acids, acylation is substantially enhanced through an internal acyl transfer mechanism. Acylation initially occurs on the phenolic oxygen atom, enabled by the intramolecular presence of an amine base, and is followed by an acyl transfer from the oxygen to the nitrogen atom. Fmoc-solid phase synthesis then proceeds, with significantly improved yields for peptide sequences that are difficult to assemble using standard SPPS.

After assembly the peptide is deprotected and cleaved using TFA, with concurrent removal of the Hmb backbone substituent yielding target unprotected peptide in high yields and purity. This backbone protection can also be employed to prevent aspartimide formation, and to improve solubility of protected peptides. In a recent report Hmb groups were introduced on resin-bound peptide via reductive amination, thereby avoiding the use of the more tedious N,O-bis-Fmoc (Hmb)amino acids (Nicolas et al., 1997).
There are two major limitations in the Hmb-backbone protection strategy. Firstly the internal O-to-N acyl transfer only proceeds well for non-hindered cases. When a β-branched amino acid has to be coupled to a Nα-Hmb residue other than glycine several hours of heating (80° C.) is required for the rearrangement to proceed. Secondly, this group is only compatible with Fmoc chemistry, and not with the often-preferred BOC SPPS, due to its TFA lability.
The Hmb methodology has demonstrated that backbone substitution can alleviate sequence related assembly problems for Fmoc chemistry. However, for hindered cases it creates additional problems of its own. The methodology would benefit significantly from the development of a more acid stable auxiliary that would allow a faster intramolecular acyl transfer (that does not require heating) and improved assembly of difficult sequences using either Fmoc or Boc SPPS.
This Hmb-backbone substitution approach has led to the recent development of backbone amide linkers (BAL) (Jensen et al., 1998), as shown in Scheme 7.

A tris-alkoxybenzyl unit is employed to link a peptide via the backbone amide nitrogen atom to a solid support. The link is cleaved by simple TFA treatment at the end of the synthesis. This linking strategy, in contrast to most other such strategies, does not make use of the C-terminal carboxylic acid, and can, at least in theory, be used on any amide bond. It is thus especially useful for synthesis of C-terminaly modified peptides or for the on-resin synthesis of head-to-tail cyclic peptides. As for the Hmb group, the first limitation lies in the difficulties of acylating the secondary amine to form the “linked” amide bond. A second problem is that standard Fmoc SPPS leads to almost complete diketopiperazine formation at the dipeptide stage. Special protection strategies need to be employed to avoid this problem.
The most valuable auxiliary strategies for peptide ligation, cyclisation or difficult peptide sequence assembly generate unmodified peptide backbones in the final product. There are three critical features in these auxiliary strategies: introduction, acylation and removal, as illustrated in Scheme 8. The prior art strategies have been successfully applied in a limited number of cases. However, applications of these strategies are severely limited by the difficulties encountered in the acyl transfer step and/or the final auxiliary removal. Often the acyl transfer is very slow, or does not proceed at all.

It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.
There are at least three requirements needed to make the auxiliary approach more versatile:
1. allow generic introduction of the auxiliary at the N atom,
2. allow more effective acylation of the nitrogen atom, and
3. allow removal of the auxiliary after acylation.
This combination of requirements severely limits the design of novel auxiliaries.
We have surprisingly found that a modification of the molecular fragment that links an oxygen or sulfur atom to the nitrogen atom has a strong accelerating effect on the acylation rate of the nitrogen atom, in contrast to prior art examples. In a particularly preferred embodiment, the modification further allows photolytic cleavage of the covalent bond between the acylated nitrogen atom and the remaining molecular fragment that connects the nitrogen atom with the oxygen or sulfur atom.
This approach is particularly useful in the field of peptide synthetic chemistry for applications such as formation of small cyclic peptides, formation of large peptides through native ligation of smaller peptide fragments, synthesis of “difficult” peptides, and backbone-linking to a solid support. The prior art methods are often not effective, ie. they only work for a small number of examples, and thus are not generic. This invention provides a more versatile approach for the synthesis of small cyclic peptides, ligation of peptide segments, backbone protection and linkage of peptide to resin during solid phase peptide assembly.